Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Enhanced Stability of Tyrosine Phenol-Lyase from Symbiobacterium toebii by DNA Shuffling

  • Kim, Jin-Ho (Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Song, Jae-Jun (Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Bong-Gyun (Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Sung, Moon-Hee (BioLeaders Corporation, Department of Bio & Nanochemistry, Kookmin University) ;
  • Lee, Sang-Chul (Korea Research Institute of Bioscience and Biotechnology (KRIBB))
  • Published : 2004.02.01
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Abstract

Tyrosine phenol-lyase (TPL) is a useful enzyme for the synthesis of pharmaceutical aromatic amino acids. In the current study, sequential DNA shuffling and screening were used to enhance the stability of TPL. Twenty-thousand mutants were screened, and several improved variants were isolated. One variant named A13V, in which the $13^{th}$ amino acid alanine was substituted by valine, exhibited a higher temperature and denaturant stability than the wild-type TPL. The purified mutant TPL, A13V, retained about 60% of its activity at $76^\circ{C}$, whereas the activity of the wild-type TPL decreased to less than 20% at the same temperature. Plus, A13V exhibited about 50% activity with 3 M urea, while the wild-type TPL lost almost all its catalytic activity, indicating an increased denaturant tolerance in the mutant A13V. It is speculated that the substitution of Val for the Ala in the $\beta$-strand of the N-terminal arm was responsible for the heightened stabilization, and that the current results will contribute to further research on the structural stability of TPL.

Keywords

References

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